Medium composition

ABSTRACT

Disclosed are a medium composition for culturing Clostridium botulinum comprising a potato peptone, a yeast extract and glucose, and a method of preparing a botulinum toxin using the same. The medium excludes animal-derived products and major allergens, and enables the botulinum toxin-producing strain to reach the maximum growth level within a short time, thereby providing effects of shortening the production time and reducing the production cost in the botulinum toxin production process.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a medium composition for culturingClostridium botulinum and a method for preparing a botulinum toxin usingthe same, and more specifically, to a medium composition for culturingClostridium botulinum that comprises a potato peptone, a yeast extractand glucose and is capable of effectively shortening the culture time ofClostridium botulinum while excluding animal-derived products and majorallergens, and is thus, and a method for preparing a botulinum toxinusing the same.

Description of the Related Art

Botulinum toxin is a neurotoxic protein produced by bacteria such asClostridium butyricum, Clostridium baraffi, and Clostridium botulinum.Botulinum toxin blocks neuromuscular transmission and causesneuroparalytic diseases in humans and animals. In particular, botulinumtoxin type A is known to be very fatal to humans. In addition tobotulinum toxin type A, six other botulinum toxins types B, C1, D, E, F,G and H have been identified. Each botulinum toxin type can bedistinguished by a corresponding type-specific antibody, and there is adifference in the severity of the paralysis caused thereby and theanimal species affected thereby.

The molecular weight of the botulinum toxin protein molecule is about150 kD, including a light chain of about 50 kD and a heavy chain ofabout 100 kD conjugated thereto. However, botulinum toxin released fromClostridium bacteria is released in the form of a complex of a 150 kDtoxin protein with at least one non-toxin protein. For example,botulinum toxin is released as 900 kD, 500 kD and 300 kD complexes.

Botulinum toxin may be very fatal to humans, but botulinum toxin hasrecently been developed to treat a variety of symptoms includingneuromuscular disorders characterized by skeletal muscle hyperactivity.For example, Botox® is a trademark of botulinum toxin A commerciallydeveloped by Allergan, Inc., which is used to alleviate or treatblepharospasm, strabismus, cervical dystonia and glabella (facial)wrinkles, and research is underway to develop applications suitable forother serotypes and clinically utilize the serotypes.

Botulinum toxins for clinical use are generally isolated from cellcultures. Conventionally, botulinum toxins have been mainly isolatedthrough culture, fermentation and purification processes usinganimal-derived products. However, when botulinum toxin is produced usinganimal-derived products, there is a concern in that, when the botulinumtoxin is administered to a patient, various pathogens or infectioussubstances derived from the animals may also be administered thereto.For example, prions may be incorporated in contained in a producedbotulinum toxin composition. The prion is a type of disease infectionfactor completely different from bacteria, viruses, fungi and parasites.The animals including humans infected with the prion undergo perforationof the brain like a sponge and death of nerve cells, resulting in lossof the corresponding brain function. Prions can generate an abnormalconformational isoform from the same nucleic acid sequence producing anormal protein, and infectivity is present during the “recruitmentreaction” in which normal isomers become prion protein isoforms in thepost-translational stage. Normal intrinsic cellular proteins inducemisfolding into pathogenic prion structures. Creutzfeldt-Jakob diseaseis a rare neurodegenerative disease of human transmissable spongiformencephalopathies and the infectious substance is an abnormal isoform ofthe prion protein. Subjects with the Creutzfeldt-Jakob disease may beaggravated into akinetic mutism from the health condition within sixmonths. Thus, administration with a pharmaceutical compositioncontaining a biological agent such as botulinum toxin obtained usinganimal-derived products has a risk of causing a prion-mediated diseasesuch as Creutzfeldt-Jakob disease.

In order to remove the risk, for example, attempts have been made toexclude animal-derived ingredients from a culture medium in the processof producing a botulinum toxin. As a representative example, AllerganInc. devised a method of conducting fermentation in a medium containingsoybeans as a plant-derived ingredient, instead of an animal-derivedingredient (Korean Patent Laid-open No. 10-2006-0102330), but the methodhas a problem in that the strain should be cultured for a long time toproduce a sufficient amount of botulinum toxin.

Food allergy is an abnormal response to a digested food triggered by theimmune system. Food allergy generally involves symptoms causing seriousdiscomforts in everyday life such as hives, angioedema and atopicdermatitis, and may result in anaphylaxis, which is a serious anddangerous allergic reaction that has the potential to becomelife-threatening. The eight major causes of food allergy published bythe FDA are milk, eggs, fish, crustaceans, nuts, peanuts, wheat andsoybeans, which account for 85 to 90% of the total causes.

Accordingly, as a result of extensive efforts to develop a componentcapable of effectively culturing a botulinum toxin-producing strainwhile replacing a conventional medium containing an animal-derivedingredient and excluding possible allergens, the present inventors foundthat, when culturing Clostridium butyricum in a medium compositioncontaining a potato peptone, a yeast extract and glucose, the culturespeed can be significantly improved so that Clostridium butyricum canreach a maximum growth level within a very short time. Based on thisfinding, the present invention has been completed.

SUMMARY OF THE INVENTION

Therefore, the present invention has been made in view of the aboveproblems, and it is an object of the present invention to provide amedium composition capable of improving the growth speed of a botulinumtoxin-producing strain, compared to a conventional medium composition,as a composition excluding an animal-derived product and a majorallergen.

In accordance with an aspect of the present invention, the above andother objects can be accomplished by the provision of a mediumcomposition for culturing Clostridium botulinum comprising a potatopeptone, a yeast extract and glucose, wherein the composition is free ofanimal-derived ingredients and allergens.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 shows the result of normal distribution analysis based on resultsof factor experiments conducted within eight concentration ranges oncomponents according to the present invention;

FIG. 2 shows the result of pareto analysis based on results of factorexperiments conducted within eight concentration ranges on componentsaccording to the present invention;

FIG. 3 shows the result of model suitability analysis based on theresults of factor experiments, center point experiments and axial pointexperiments on the components according to the present invention;

FIG. 4 shows the result of surface analysis based on the results offactor experiments, center point experiment and axial point experimentsconducted on the components according to the present invention;

FIG. 5 shows the result of determination of the optimal ratio based onthe results of surface analysis on the components according to thepresent invention;

FIG. 6 shows the result of determination of the absorbance according tothe toxin level and strain growth with regard to a variable glucoseconcentration in the optimal ratio according to the present invention;and

FIG. 7 shows the result of determination of the absorbance according tothe toxin level and strain growth over time, while culturing Clostridiumbotulinum in the optimized medium composition of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as appreciated by those skilled in the field towhich the present invention pertains. In general, the nomenclature usedherein is well-known in the art and is ordinarily used.

In the present invention, it was identified that, when the Clostridiumbotulinum strain was cultured in a culture medium containing a potatopeptone, a yeast extract and glucose, as a culture medium which is freeof animal-derived ingredients and allergens, the strain reached themaximum growth level within a very short time and the culture speed wassignificantly improved.

Accordingly, in one aspect, the present invention is directed to amedium composition for culturing Clostridium botulinum, comprising apotato peptone, a yeast extract and glucose.

The botulinum toxin-producing strain used in the present invention maybe Clostridium botulinum or a variant thereof, most preferablyClostridium botulinum type A, NCTC13319, but is not limited thereto. Itwill be apparent to those skilled in the art that any strain capable ofproducing botulinum toxins can be used.

The present invention is characterized in that the medium compositioncomprises 2 to 5% (w/v) of the potato peptone, 0.5 to 2% (w/v) of theyeast extract and 0.75 to 1.5% (w/v) of the glucose, most preferably,the medium composition comprises 3% (w/v) of the potato peptone, 1%(w/v) of the yeast extract and 1% (w/v) of the glucose, but is notlimited thereto.

The composition ratio of the ingredients as described above is acomposition ratio of the components obtaining 90% of a toxin productionlevel, obtained through a surface analysis method based on theoptimization concentration setting program of the minitab program, whenthe toxin production level in a medium composition containing 3% (w/v)of a potato peptone, 1% (w/v) of a yeast extract and 1% (w/v) ofglucose, which is the best mode of the composition ratio of thecomponents of the present invention, is assumed to be 100%. That is, thetoxin production level corresponding to 90% of the toxin productionlevel obtained with the medium composition containing 3% (w/v) of thepotato peptone, 1% (w/v) of the yeast extract and 1% (w/v) of theglucose can be obtained when 2 to 5% (w/v) of the potato peptone, 0.5 to2% (w/v) of the yeast extract and 0.75 to 1.5% (w/v) of the glucose arecontained in the medium composition.

Meanwhile, in the present invention, it was identified that, whenculturing a Clostridium botulinum strain using the medium composition,the culture speed increases and the recovery timing can thus besignificantly shortened in the process of producing the botulinum toxin.That is, in the culture of Clostridium botulinum using soybean peptone,Clostridium botulinum exhibits the maximum growth level after 24 hours.On the other hand, Clostridium botulinum according to the presentinvention exhibits the maximum growth level in about 14 to 18 hours.Regarding the toxin recovery timing, in the case of the conventionalculture of Clostridium botulinum, the maximum toxin production wasobtained after culture for at least 48 hours culture, whereas in thepresent invention, the maximum toxin production was obtained within 48hours, that is, at about 37 hours culture (see FIG. 7).

Accordingly, in another aspect, the present invention is directed to amethod of preparing a botulinum toxin comprising (a) producing abotulinum toxin by culturing Clostridium botulinum in a mediumcomposition for culturing Clostridium botulinum comprising a potatopeptone, a yeast extract and glucose \, and (b) recovering the botulinumtoxin.

In the present invention, the botulinum toxin may be recovered within 48hours after initiation of culture in step (b). In another embodiment,the botulinum toxin may be recovered within 40 hours after initiation ofculture in step (b).

In the present invention, the term “excluding an animal-derivedingredient” means substantially excluding an animal-derived ingredientor substantially excluding an animal protein, and specifically meansthat a blood-derived, blood-pooled and other animal-derived product orcompound is absent or substantially absent. The term “animal” meansmammals (such as humans), birds, reptiles, fish, insects, spiders orother animal species. The “animal” does not include microorganisms suchas bacteria. Thus, a medium or method excluding an animal product, or amedium or method substantially excluding an animal product, which fallswithin the scope of the present invention, may include botulinum toxinsor Clostridium botulinum bacteria. For example, a method excluding ananimal product or a method substantially excluding an animal productmeans a method that substantially excludes, essentially excludes, orcompletely excludes an animal-derived protein such as an immunoglobulin,meat digest, meat byproduct, and milk or dairy product or digest. Thus,examples of the method excluding an animal product include a method(such as bacterial culture or bacterial fermentation method) excludingmeat and dairy products or meat and dairy byproducts.

As used herein, the term “botulinum toxin” means not only a neurotoxinproduced by Clostridium botulinum but also a botulinum toxin (or lightor heavy chain) produced recombinantly by a non-Clostridium botulinumspecies. As used herein, the term “botulinum toxin” refers to botulinumtoxin subtypes A, B, C, D, E, F, G and H (Weller C (15 Oct. 2013). “NewBotulinum Toxin Deemed Deadliest Substance Ever: Sniffing 13-Billionthsof a Gram Can Kill”. Medical Daily.) and G. As used herein, botulinumtoxin also includes both a botulinum toxin complex (i.e., 300, 600 and900 kDa complexes) as well as a pure botulinum toxin (i.e., about 150kDa). The term “pure botulinum toxin” is defined as a botulinum toxinisolated or substantially isolated from other proteins includingproteins forming the botulinum toxin complex. The pure botulinum toxinmay have a purity of 95% or higher, preferably 99% or higher.

The present invention provides a medium that includes at least reducedlevels of animal or dairy by-products, substantially free of animal ordairy by-products. The term “animal or dairy by-product” means acompound or a combination of compounds prepared in or by animal(excluding bacteria) cells, in vivo or in vitro. Preferred non-animalsources of medium ingredients such as proteins, amino acids and nitrogeninclude plants, microorganisms (such as yeasts) and synthetic compounds.

The medium according to the present invention includes, but is notlimited to, a medium for fermentation of a small or large amount ofClostridium botulinum, a medium for the growth and culture ofClostridium botulinum used to inoculate into a seed (primary) medium anda fermentation (secondary) medium, and a medium used for long-termstorage of Clostridium botulinum cultures (e.g., stock cultures).

As a specific preferred embodiment of the present invention, the mediumfor growth of Clostridium botulinum and production of botulinum toxinsmay include a potato-derived ingredient, preferably a potato peptone,replacing an animal-derived ingredient.

The present invention provides a method for growing Clostridiumbotulinum capable of maximizing the production of botulinum toxinswithin the shortest time using a medium substantially free of ananimal-derived ingredient. Clostridium botulinum can be grown using amedium composition containing a potato peptone, instead of ananimal-derived ingredient.

In a preferred embodiment of the present invention, the growth ofClostridium botulinum is carried out in two steps (i.e., seed growth andfermentation). Both of these steps are preferably conducted in ananaerobic environment. The seed growth is generally used to “scale-up”the amount of microorganisms from the stored culture. The purpose of theseed growth is to increase the amount of microorganisms that can be usedfor fermentation. In addition, the seed growth allows the relativelydormant microorganisms in the stored culture to rejuvenate and grow intoan actively growing culture. In addition, the volume and amount ofviable microorganisms used to inoculate into the fermentation medium canbe more precisely controlled in a more actively growing culture than inthe stored culture. Therefore, growth of seed culture for inoculationinto a fermentation medium is preferred. In addition, several successivesteps including growth in a seed medium to scale-up the amount ofClostridium botulinum for inoculation into a fermentation medium can beused. The growth of Clostridium botulinum in the fermentation step mayalso be carried out by direct inoculation from the stored medium.

In the fermentation step, a part or entirety of the seed mediumcontaining Clostridium botulinum from the seed growth product can beused for inoculation into the fermentation medium. Preferably, about 1to 10% of the seed medium containing Clostridium botulinum from the seedgrowth product is used for inoculation into the fermentation medium. Thefermentation is used to produce the largest amount of microorganisms ina large-scale anaerobic environment.

The botulinum toxin contained in the culture of the strain cultured bythe method may be isolated and purified using protein purificationmethods known to those skilled in the art of protein purification.

The growth of Clostridium botulinum can be carried out in one or moresteps. Preferably, growth proceeds in two steps. In the first step, seedgrowth, Clostridium botulinum is suspended in the medium compositionaccording to the invention and cultured at 34±1° C. under an anaerobicenvironment for 10 to 24 hours. Preferably, seed growth is carried outfor about 14 hours. It is also preferred that growth in the seed mediumat any step does not cause cell autolysis prior to inoculation of thefinal growth product in the seed medium with the fermentation medium.

Subsequently, in order to further grow Clostridium botulinum and recoverthe botulinum toxin, the second step, fermentation, is performed byinoculating the medium composition of the present invention using partor all of the seed-grown medium. After inoculation, Clostridiumbotulinum is also cultured at 34±1° C. under an anaerobic environmentfor about 4 days and the growth thereof is monitored by measuring theoptical density (OD) of the medium. Preferably, in the fermentation stepusing the medium composition according to the present invention, themaximum growth level is obtained after about 14 and 18 hours, the cellsare lysed and the OD value decreases, whereby the botulinum toxin isrecovered from the Clostridium botulinum within 48 hours, morepreferably within 40 hours, after incubation, in the fermentation step.

In a preferred embodiment of the present invention, when the culture ofClostridium botulinum is stored at 4° C. for long-term storage ofClostridium botulinum and coming inoculation of the seed medium,Clostridium botulinum is preferably stored in a storage medium (3% ofpotato peptone, 1% of yeast extract, of 1% glucose and 25% of glycerol)in order to keep the medium substantially free of animal by-productsthroughout the production of botulinum toxin.

EXAMPLE

Hereinafter, the present invention will be described in more detail withreference to examples. However, it will be obvious to those skilled inthe art that these examples are provided only for illustration of thepresent invention and should not be construed as limiting the scope ofthe present invention.

Example 1: Sample Preparation and Bacterial Culture 1-1. SamplePreparation

The botulinum strain used in the present invention is Clostridiumbotulinum type A, NCTC13319 and the samples used to prepare the media ofthe experimental group and the control group are as follows: Phytonepeptone (BD, 211906), Tryptone (BD, 211705), Yeast extract (BD, 212750),Potato peptone E210 (Organotechnie, 19425), Pea peptone A482(Organotechnie, A1275), Plant peptone E1 (Organotechnie, 19025), Selectphytone UF (BD, 210931), Peptone from soybean (Sigma, 70178), Hy-Soy(Kerry, 5X59022), Hy-peptone (Kerry, 5X01111), Soy peptone A2SC(Organotechnie, 19649), Soy peptone A3SC (Organotechnie, 19685), Soypeptone E110 (Organotechnie, 19885), Soy peptone K200 (Organotechnie,19749), Glucose (Sigma, G5767), Sodium thioglycolate (Sigma, T0632),Purified water (Ultrapure water or water with quality equal to or higherthan ultrapure water).

1-2. Bacterial Culture

One vial containing the Clostridium botulinum strain stored in acryogenic freezer was thawed in a 37° C. incubator for 30 minutes. TheClostridium botulinum strain was homogenized 3 to 5 times in BSC, 0.1 mLof the homogenized Clostridium botulinum strain was inoculated into amedium and were then cultured at 34±1° C. in an anaerobic incubator.After the culture, OD₆₀₀ was measured using a spectrophotometer.

1-3. Toxin Level Measurement

The level of toxin was measured by the test method of BotulinumNeurotoxin Type A DuoSet ELISA (R&D system) controlled according to theinstructions of the manufacturer. 100 μL of a capture antibody preparedby dilution in PBS was seeded into each well of a 96-well microplate andcoated for 16 hours or more, and washing each well with a wash bufferwas repeated three times in total. Each well was blocked with a reagentdiluent and was then washed 3 times in total with a wash buffer.

After the culture was completed, the culture solution was sterilizedusing a 0.2 μm syringe filter, diluted, added in an amount of 100 μL toeach well and allowed to react for 2 hours, and then washing each wellwith a wash buffer was repeated a total of three times. 100 μL of adetection antibody dilution was added to each well and allowed to reactfor 2 hours, and washing each well with a wash buffer was repeated atotal of 3 times. 100 μL of a streptavidin-HRP dilution was added toeach well and allowed to react for 20 minutes, and washing each wellwith a wash buffer was repeated a total of 3 times. 100 μL of asubstrate solution was added to each well and allowed to react for 20minutes, and 50 μL of a stop solution was added to each well andappropriately shaken to stop the enzyme reaction. 450 nm absorbance wasmeasured in each well using a microplate reader.

Example 2. Selection of Peptone for TPYG Medium Replacement

The TPYG medium containing a casein peptone, a soy peptone, a yeastextract, glucose and sodium-thioglycolate, which has been commonly usedas a conventional medium for producing a botulinum toxin, was attemptedto replace with a PYG medium including a peptone, a yeast extract,glucose and sodium-thioglycolate.

Regarding the peptone, a variety of candidates including plant peptonewere selected and a plant-derived medium having a similar toxinproduction to a TYPG medium was primarily selected among a soy-derivedmedium and other plant-derived medium.

2-1. Comparative Example: Preparation of TPYG Medium

The phytone peptone, tryptone, yeast extract and sodium thioglycolatewere weighed as 2 g, 1 g, 1 g, and 0.1 respectively, using a weighingdish, and 50 mL of purified water was added to a 250 mL beaker, and theweighed medium ingredients were added thereto and stirred until theingredients were dissolved completely. The pH was adjusted to 7.2 with1N NaOH, the medium of the beaker was transferred to a 100 mL masscylinder, purified water was added thereto to adjust the final liquidlevel to 95 mL, and then the prepared medium and a magnetic bar weretransferred to a 100 mL glass bottle and the lid was closed.

Glucose was weighed as 1 g using a weighing dish, and the weighedglucose was put into a 15 mL conical tube containing approximately 3 mLof purified water and vortexed to dissolve sufficiently the glucose, andthen purified water was added to the conical tube to adjust the finalsolution level to 5 mL.

The lid of the glass bottle was slightly opened, and the glass bottlewas wrapped with foil and sterilized in an autoclave at 121° C. for 20minutes. The sterilized medium and glucose were sufficiently cooled inBSC and then glucose was transferred to the medium and mixed.

2-2. Experimental Example: Preparation of PYG Medium

The 12 medium ingredients shown in Table 1 were weighed and dissolved in50 mL of purified water, the pH was adjusted to 7.2 with 1N NaOH,purified water was added to adjust the final liquid level as shown inTable 1, and the prepared medium and a magnetic bar were transferred toa 100 mL glass bottle and the lid thereof was closed.

TABLE 1 Ingredient (g) Medium Yeast Final liquid level name Peptoneextract (mL) PYG1 Phytone peptone 3 1 95 mL PYG2 Potato peptone E210 395 mL PYG3 Pea peptone A482 3 95 mL PYG4 Plant peptone E1 3 95 mL PYG5Select phytone UF 3 95 mL PYG6 Peptone from soybean 3 95 mL PYG7 Hy-soy3 95 mL PYG8 Hy-peptone 3 95 mL PYG9 Soy peptone A2SC 3 95 mL PYG10 Soypeptone A3SC 3 95 mL PYG11 Soy peptone E110 3 95 mL PYG12 Soy peptoneK200 3 95 mL

Glucose was weighed as 13 g with a weighing dish and the weighed glucosewas dissolved in 65 mL of purified water. The prepared glucose solutionwas transferred to a 100 mL glass bottle and the lid was closed.

The prepared medium and glucose were sterilized at 121° C. for 20minutes, the sterilized medium and glucose were sufficiently cooled inBSC, and 5 mL of the glucose solution was transferred to each medium andmixed.

2-3. Pepton Selection

The Clostridium botulinum strain was cultured in the medium prepared inExample 2-1 or Example 2-2 in the same manner as in Example 1-2, theOD₆₀₀ value was determined at the end of the culture, and the toxinproduction level was determined by ELISA in the same manner as in 1-3.

TABLE 2 Medium name TPYG PYG1 PYG2 PYG3 PYG4 OD₆₀₀ 0.437 0.463 0.3850.309 0.013 Toxin level 22.7 21.5 25.4 2.7 15.6 (mg/L) Toxin level 10094.7 111.9 11.9 68.7 (relative value) Medium name TPYG PYG5 PYG6 PYG7PYG8 OD₆₀₀ 0.447 0.360 0.372 0.414 0.532 Toxin level 28.8 27.0 16.6 18.518.5 (mg/L) Toxin level 100 93.8 57.6 64.2 64.2 (relative value) Mediumname TPYG PYG9 PYG10 PYG11 PYG12 OD₆₀₀ 0.396 0.147 0.526 1.980 2.120Toxin level 27.3 17.2 8.7 15.9 7.2 (mg/L) Toxin level 100 63.0 31.9 58.226.4 (relative value)

As a result, OD₆₀₀ and toxin production level after completion of theculture were shown in Table 2, and three mediums (PYG1, PYG2, and PYG5)showed similar toxin levels and maximum strain dissolution, and thusOD₆₀₀ of less than 1, compared to the TPYG medium. These results wereverified again through repeated tests. As can be seen from Table 3,PYG1, PYG2 and PYG5 showed similar toxin production levels to TPYG inthe repeated tests as well. Among them, the PYG2 medium excluding ananimal ingredient and a major allergen was used to obtain the bestmedium composition.

TABLE 3 Medium name TPYG PYG1 PYG2 PYG5 OD₆₀₀ 0.541 0.514 0.650 0.433Toxin level (mg/L) 27.4 28.4 24.7 22.3 Toxin level 100 103.6 90.1 81.4(relative value)

Example 3. Medium Composition Optimization

In order to find the optimal concentrations of potato peptone, yeastextract and glucose determined through a medium screening test, aconcentration range test was conducted through DoE and the optimumcomposition was determined based on the results of factor experiments,center point experiments and axial point experiments.

3-1. Factor Experiment

Factor experiments were conducted using medium ingredients includingingredients with eight concentration ranges as shown in Table 4. Themedium was prepared in the same manner as 2-2 using the ingredientsincluded in Table 4, and the Clostridium botulinum strain was culturedin the prepared medium in the same manner as in Example 1-2, the OD₆₀₀value at the end of the culture was determined and the toxin productionlevel was determined through ELISA in the same manner as in 1-3.

TABLE 4 Ingredient Final Potato peptone Yeast Glucose liquid levelMedium name E210 (g) Extract (g) (mL) (mL) Factor 2 0.5 10 90 experiment1 Factor 5 0.5 2.5 97.5 experiment 2 Factor 2 2 2.5 97.5 experiment 3Factor 5 2 10 90 experiment 4 Factor 2 0.5 2.5 97.5 experiment 5 Factor5 2 2.5 97.5 experiment 6 Factor 5 0.5 10 90 experiment 7 Factor 2 2 1090 experiment 8

As a result, OD₆₀₀ and toxin levels were determined as shown in Table 5.As a result of the normal distribution analysis based on the same,analysis including primary and secondary interactions while excludingternary interactions showed that the remaining factors except for thepeptone*glucose interaction in the first-order and second-order terms ofpeptone, yeast and glucose are significant in the toxin production level(FIG. 1), and Pareto analysis also showed the same results as the normaldistribution analysis (FIG. 2).

TABLE 5 Medium name Medium composition OD₆₀₀ Toxin level (mg/L) FactorPotato peptone 2% 4.560 20.0 experiment 1 Yeast extract 0.5% Glucose 2%Factor Potato peptone 5% 0.805 3.0 experiment 2 Yeast extract 0.5%Glucose 0.5% Factor Potato peptone 2% 0.447 3.0 experiment 3 Yeastextract 2% Glucose 0.5% Factor Potato peptone 5% 7.600 9.6 experiment 4Yeast extract 2% Glucose 2% Factor Potato peptone 2% 0.267 5.2experiment 5 Yeast extract 0.5% Glucose 0.5% Factor Potato peptone 5%0.838 4.5 experiment 6 Yeast extract 2% Glucose 0.5% Factor Potatopeptone 5% 6.350 14.3 experiment 7 Yeast extract 0.5% Glucose 2% FactorPotato peptone 2% 5.080 13.0 experiment 8 Yeast extract 2% Glucose 2%

3-2. Center Point Experiment

Potato peptone E210, yeast extract and sodium thioglycolate for a mediumfor center point experiments were weighed as 24.5 g, 8.75 g, and 0.7 gusing a weighing dish, respectively, 550 mL of purified water was put ina 1 L beaker, and the weighted ingredients were stirred therein untilthey were completely dissolved. The pH was adjusted to 7.2 with 1N NaOH,the medium of the beaker was transferred to a 1 L mass cylinder,purified water was added thereto to adjust the final liquid level to656.25 mL, and then the final liquid was transferred to a 1 L beaker andstirred for about 5 minutes. The medium was transferred in 93.75 mL toeach of a total of six 100 mL glass bottles, a magnetic bar was injectedinto the glass bottle and the lid was closed, and each medium wasdesignated as any one of center points 1 to 6.

Glucose was weighed as 8 g using a weighing dish and the weighed glucosewas completely dissolved in 40 mL of purified water.

The prepared medium and glucose were sterilized at 121° C. for 20minutes, the sterilized medium and glucose were sufficiently cooled inBSC, and 6.25 mL of the glucose solution was transferred and mixed witheach medium.

The Clostridium botulinum strain was cultured in the medium thusprepared in the same manner as in Example 1-2, the OD₆₀₀ value wasdetermined at the end of the culture, and toxin production level wasdetermined by ELISA in the same manner as in 1-3.

TABLE 6 Medium name Medium composition OD₆₀₀ Toxin level (mg/L) Centerpoint 1 Potato peptone 3.5% 3.360 20.2 Yeast extract 1.25% Glucose 1.25%Center point 2 Potato peptone 3.5% 1.630 21.1 Yeast extract 1.25%Glucose 1.25% Center point 3 Potato peptone 3.5% 4.810 16.4 Yeastextract 1.25% Glucose 1.25% Center point 4 Potato peptone 3.5% 4.30016.2 Yeast extract 1.25% Glucose 1.25% Center point 5 Potato peptone3.5% 4.830 15.9 Yeast extract 1.25% Glucose 1.25% Center point 6 Potatopeptone 3.5% 5.010 15.1 Yeast extract 1.25% Glucose 1.25%

The center point experiment of one concentration was repeated 6 times,and the results were shown in Table 6. When determining the presence orabsence of the curve of the graph based on the results of the centerpoint experiments, the p-value was 0.001, which is less than 0.05, whichis a criterion that is determined to be significant. This means that thecurve is present, and an additional test to conduct surface analysis wasperformed.

TABLE 7 Analysis of Variance for toxin (coded units) Source

Seq SS Adj SS Adj MS F P

3

1

1

1

3

1

1

1

Curvature 1 213.62

213.62

213.6

2

1

1

Total 9

indicates data missing or illegible when filed

3-3. Axial Point Experiment

Six medium ingredients for a medium for the axial point experiment wereweighed as shown in Table 8 and dissolved in 50 mL of purified water,the pH was adjusted to 7.2 with 1N NaOH, and purified water was addedthereto to adjust the final liquid level as shown in Table 8. Eachprepared medium and magnetic bar were transferred to a 100 mL glassbottle and the lid was closed.

TABLE 8 Ingredient Final Potato peptone Yeast Glucose liquid levelMedium name E210 (g) Extract (g) (mL) (mL) Axial point 1 3.5 1.25 12.587.5 Axial point 2 1 1.25 6.25 93.75 Axial point 3 6 1.25 6.25 93.75Axial point 4 3.5 0.025 6.25 93.75 Axial point 5 3.5 2.5 6.25 93.75Axial point 6 3.5 1.25 0.125 99.875

Glucose was weighed as 8 g using a weighing dish and the weighed glucosewas completely dissolved in 40 mL of purified water. The prepared mediumand glucose were sterilized in an autoclave at 121° C. for 20 minutes,the sterilized medium and glucose were sufficiently cooled in BSC andthen the glucose solution was added in an amount shown in Table 8 toeach medium.

The Clostridium botulinum strain was cultured in the medium thusprepared in the same manner as in Example 1-2, the OD₆₀₀ value wasdetermined at the end of the culture, and toxin production level wasdetermined by ELISA in the same manner as in 1-3. As a result, the axialpoint value was determined as shown in Table 9.

TABLE 9 Medium name Medium composition OD₆₀₀ Toxin level (mg/L) Axialpoint 1 Potato peptone 3.5% 5.53 7.2 Yeast extract 1.25% Glucose 2.5%Axial point 2 Potato peptone 1% 2.550 12.8 Yeast extract 1.25% Glucose1.25% Axial point 3 Potato peptone 6% 0.416 20.0 Yeast extract 1.25%Glucose 1.25% Axial point 4 Potato peptone 3.5% 2.240 8.1 Yeast extract0.025% Glucose 1.25% Axial point 5 Potato peptone 3.5% 0.447 21.2 Yeastextract 2.5% Glucose 1.25% Axial point 6 Potato peptone 3.5% 0.986 0.3Yeast extract 1.25% Glucose 0.025%

3-4. Optimal Concentration Analysis Through Surface Analysis

The surface analysis was conducted based on the results of the factorexperiment, center point experiment and axial point experiment. For themodel suitability analysis, normal probability plots, histograms, plotsof residuals versus the fitted values and plots of residuals versus theorder were analyzed. The normal probability plot was determined as astraight line, the histogram appeared in the form of a normaldistribution, and plots of residuals versus the fitted values and plotsof residuals versus the order had no pattern. Based on these, theselected model was determined suitable (FIG. 3).

The result was analyzed using surface analysis to determine the culturecomposition, the result is shown in FIG. 4, and the optimal ratio wascalculated based on the result as shown in FIG. 5 and the optimalconcentration obtained from the optimal ratio was summarized in Table10.

TABLE 10 Ingredient name Potato peptone Yeast extract Glucose Optimumconcentration 3% 1% 1.5% (%)

3-5. Determination of Optimum Composition Ratio

The optimal concentration set through the surface analysis method wasbased on the toxin production level, and the final target was to achievea composition having the highest toxin production level and theconcentration of OD₆₀₀ after incubation of 1 or less. Thus, the optimumculture composition was obtained by changing the glucose concentrationconsidered to have the greatest effects on toxin production level andOD₆₀₀ to 0.75, 1, 1.25, 1.5%, and the results are shown in Tables 11 andFIG. 6. The optimal medium composition was determined as 3% potatopeptone, 1% yeast extract and 1% glucose.

TABLE 11 Toxin level Medium name Medium composition OD₆₀₀ (mg/L) 0.75%Potato peptone 3% 0.343 1.500 Yeast extract 1% Glucose 0.75%   1% Potatopeptone 3% 0.877 24.72 Yeast extract 1% Glucose 1% 1.25% Potato peptone3% 3.230 18.95 Yeast extract 1% Glucose 1.25%  1.5% Potato peptone 3%6.580 10.65 Yeast extract 1% Glucose 1.5%

Example 4. Strain Culture Experiment According to Optimum CultureComposition

One vial containing the Clostridium botulinum strain stored in acryogenic freezer was thawed in a 37° C. incubator for 30 minutes. TheClostridium botulinum strain was homogenized 3 to 5 times in BSC, 1 mLof the homogenized Clostridium botulinum strain was inoculated into 100mL of a PYG medium in a 2L cell bag and was then cultured at 34±1° C. inan incubator (WAVE25). 100 mL of the strain culture was inoculated into5 L of a PYG medium before the strain autolysis between 10 to 24 hours.Then, the absorbance at a wavelength of 600 nm and the toxin level weremeasured over time. Only the culture solution was recovered from eachsample using a 0.2 μm antibacterial filter, and then the toxin level wasmeasured in the same manner as in Example 1-3.

The results of measuring the change in OD and the toxin production levelover time while culturing using the culture composition set as describedabove showed that the culture composition according to the presentinvention enables OD₆₀₀ of the strain culture to reach the maximum valuewithin about 14 to 15 hours, and thus can significantly shorten theculture time compared to a conventional Clostridium botulinum mediumcomposition, and that the maximum toxin production level was obtainedafter about 28 hours at 35° C. and after 37 hours at 33° C.

Example 5. Comparison in Culture Effect Between Optimum CultureComposition and Other Culture Compositions

The culture effect, when soy peptone and the range other than theoptimum composition ratio derived in Example 3 is used, were determined.For this purpose, each culture medium was prepared in accordance withthe composition ratio shown in Table 12 and the culture effect thereofwas determined. As a result, as can be seen from Table 13, theexperimental group was advantageous in terms of toxin production levelor cell culture time compared to Comparative Example. Specifically, inthe case of using the potato peptone in Comparative Example 1, the timetaken to reach the maximum cell mass was significantly shortened, butthe maximum toxin production at the corresponding composition ratio wasfound to be much less than the experimental group. On the other hand, inthe case of using the soy peptone in Comparative Example 2, the timetaken to reach the maximum cell mass was found to take much longer thanthe experimental group.

TABLE 12 Glucose Yeast extract Peptone Experimental group 1 1 3 (potato)Comparative Example 1 0.1 1 (potato) 1 Comparative Example 1 1 3 (soy) 2

TABLE 13 Time taken to Maximum Time taken to Maximum reach toxin reachcell mass maximum production maximum (AU) cell mass (mg/L) toxinproduction Experimental 7.92 14 h 24.8 37 h group Comparative 2.74 21 h8.52 47 h Example 1 Comparative 7.64 31 h 18.25 52 h Example 2

INDUSTRIAL AVAILABILITY

According to the present invention, botulinum toxin, which is producedby excluding animal-derived ingredients and major allergens, can preventtransmissible spongiform encephalopathy caused by the accumulation ofabnormal prion proteins, and adverse reactions caused by allergicreactions (tares, angioedema, atopic dermatitis, anaphylaxis), when thebotulinum toxin prepared by the present invention administered to thehuman body. In addition, when potato peptone is used to excludeanimal-derived ingredients and major allergens, the botulinumtoxin-producing strain can be induced to reach the maximum growth levelwithin a short time, and thus there are effects of shortening theproduction time and reducing the production cost in the botulinum toxinproduction process.

Although specific configurations of the present invention have beendescribed in detail, those skilled in the art will appreciate that thisdescription is provided to set forth preferred embodiments forillustrative purposes and should not be construed as limiting the scopeof the present invention. Therefore, the substantial scope of thepresent invention is defined by the accompanying claims and equivalentsthereto.

What is claimed is:
 1. A medium composition for culturing Clostridiumbotulinum comprising a potato peptone, a yeast extract and glucose. 2.The medium composition according to claim 1, wherein the Clostridiumbotulinum is Clostridium botulinum type A.
 3. The medium compositionaccording to claim 1, wherein the medium composition for cultureexcludes an animal-derived product and an allergen.
 4. The mediumcomposition according to claim 1, wherein the medium compositioncomprises 2-5% (w/v) of the potato peptone, 0.5-2% (w/v) of the yeastextract, and 0.75-1.5% (w/v) of the glucose.
 5. The medium compositionaccording to claim 4, wherein the medium composition comprises 3%(w/v)of the potato peptone, 1% (w/v) of the yeast extract, and 1% (w/v)of the glucose.